Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb
- University of Texas at Austin, United States
- University of Texas Health Science Center at San Antonio, United States
Abstract
The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.
Data availability
All mass spec data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided in Figure 3f, Figure 1-figure supplement 2 and Figure 3-figure supplement 2 and 4.
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RNA polymerase II ChIP with dasatinib inhibitionNCBI Gene Expression Omnibus. GSE131838.
Article and author information
Author details
Nicholas A Prescott
Funding
National Institute of General Medical Sciences (R01 GM104896)
- Yan Zhang
National Institute of General Medical Sciences (R01 RM125882)
- Jennifer Brodbelt
National Institute of Biomedical Imaging and Bioengineering (R21EB018391)
- Jennifer Brodbelt
Welch Foundation (F-1778)
- Yan Zhang
Welch Foundation (F-1155)
- Jennifer Brodbelt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jerry L Workman, Stowers Institute for Medical Research, United States
Version history
- Received: May 23, 2019
- Accepted: August 5, 2019
- Accepted Manuscript published: August 6, 2019 (version 1)
- Version of Record published: August 29, 2019 (version 2)
Copyright
© 2019, Mayfield et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb
eLife 8:e48725.
https://doi.org/10.7554/eLife.48725
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